Download Atrial natriuretic peptide (ANP)

African Journal of Biotechnology Vol. 5 (25), pp. 2534-2539, 29 December, 2006
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2006 Academic Journals
Review
Atrial natriuretic peptide (ANP)-granules: ultrastructure,
morphometry and function
Eliane Florencio Gama and Romeu Rodrigues de Souza*
Universidade São Judas Tadeu, Departamento de Anatomia Humana, São Paulo, Brasil.
Accepted 6 December, 2006
The atrial granules containing the peptide hormone, Atrial natriuretic peptide (ANP) are present in the four
regions of the atrial-auricular complex (two atria and two auricles). ANP-immunoreactivity was detected in all
granules from the four regions. Ultrastructurally, atrial myocytes show the presence of very electron dense
granules, with sparsely granular and homogeneous content, coated with a double membrane. The number of
granules is greatest in the right atrium followed by the left atrium and left auricle and right auricle, in this
order. The diameter of granules in the cardiocytes is significantly largest in the right atrium and reduced via
the left auricle to the left atrium and right auricle. These data lead to suppose that the right atrium is the one
that most synthesizes and stores the ANP. The number of ANP-granules is influenced by several
physiological conditions: temperature, dehydration and nutritional condition. The main physiological
stimulus for increased ANP release is the atrial muscle stretch, which normally occurs when extra cellular
fluid volume or blood volume is elevated. The ANP is eliminated through the atrial myocytes, via exocytosis.
Granule content is released into the extra-cellular space (extrusion). The ANP causes diuresis, natriuresis,
vasodilatation and depression of blood pressure. It is also involved in the modification of the waterelectrolyte balance.
Key words: ANP-granules, ultrastructure, morphometry.
INTRODUCTION
Previous studies from the fifties (Kisch, 1956) in guinea
pig hearts have shown the presence of specific atrial
granules which has been functionally considered as an
activator of sodium and water excretion and,
consequently, blood pressure reduction (De Bold et al.,
1981; Forssmann et al., 1984; Skepper and Navaratnam,
1988; Jiao et al., 1993; Yoshihara et al., 1998). Those
granules, in the myocites of the auricles and in the atria,
contain a peptide hormone called atrial natriuretic peptide
(ANP). The atrial and auricle walls distension under
conditions of hypervolemia or blood pressure increase
would promote an increase in the circulating ANF (Varess
and Sonnerberg, 1984). Thus far, five molecules
comprise the natriuretic peptide
*Corresponding Authors E-mail: [email protected]. Tel:
551138899196
family: Atrial natriuretic peptide (ANP), Urodilatin, Brain
natriuretic peptide (BNP), C-type natriuretic peptide
(CNP) and Dendroaspis natriuretic peptide (DNP) (Cea,
2005). Here, the ANP will be considered.
ULTRASTRUCTURE OF THE ANP-GRANULES
The electron micrographs of the atrial myocytes show the
presence of very dense electron granules, with sparsely
granular and homogeneous content, coated with a double
membrane. Among those there are others less dense,
but in a smaller quantity. Most of their granules are
located near the nuclear poles, among mitochondria,
golgi complex cisterns and rough endoplasmic reticulum,
microtubules and myofilaments. In addition to the great
number of clear vesicles near the perinuclear region,
there is a small number near the plasma membrane
(Figures 1 and 2).
Gama and Rodrigues de Souza
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DISTRIBUTION OF THE ANP-GRANULES
Using immunocytochemistry, it is possible to detect with
certainty the presence of ANP in the granules (Cantin,
1985). In the guinea pig heart ANP-immunoreactivity was
detected in all granules, from all cardiocytes in the four
regions of the atrial-auricular complex. Great amount of
gold particles related to the granules has been observed,
as well as less electron dense material near to them
(Figures 3 and 4) (Gama et al., in press). In many
species, including the guinea pig, the ANP-granules are
also present in the ventricles (Mifune et al., 1992). It was
also verified in dogs that the removal of the auricle
bilaterally eliminates the ANP release (Stewart et al.,
1992).
QUANTITATIVE STUDIES
Quantitative studies on the granules were performed in
various mammalian species (Jamieson and Palade,
1964; Cantin et al., 1979; Mifune et al., 1996), but authors do not agree on the number and diameter of the
granules in the various regions of the atrial-auricular
complex. In the rat, the larger number of granules was
found in the right atrium, whereas in the guinea pig, in
the hamster, in the rabbit and in the cat, the number of
granules was larger in the left atrium (Cantin et al.,
1979). Chapeau et al. (1985) assert that there are a
larger number of granules in the right atrium. According
to Mifune et al. (1992), imunohistochemically, the most
intensely peptide-reactive cardiocytes were localized in
the right auricle.
In a recent study, Gama et al. (in press) observed that,
although not significant, the number of granules in the
right atrium was larger than in the other regions of the
atrial-auricular complex. These results are in accordance
with those of Rinne et al. (1986) and Chiu et al. (1994) to
whom the right atrium is the greater source of ANP, not
for presenting the peptide in a greater amount, but for
responding with greater intensity to mechanical and
nervous stimulus. Gama et al. (in press) observed that
the amount of ANP-granules decrease from the right to
the left auricle and from the right to the left atrium
However, a greater amount of granules in the left atrium
in relation to the right one has been reported in guinea
pig hearts (Cantin et al., 1984). The results obtained by
Skepper et al. (1989) and Avramovitch et al. (1995)
indicate that there is no statistical difference from one
side (right) in relation to the other (left) regarding the
amount of granules in the atrial–auricular complex.
Results showing that the right auricle is functionally more
active than the left one have been reported (Varess and
Sonnenberg, 1984; Skepper et al., 1989; Stewart et al.,
1992). It was also verified in dogs that the removal of the
auricle bilaterally eliminates the ANP release (Stewart et
al., 1992). It is possible, that these different results are
due to different methods used.
Figure 1. Electron micrograph of right atrium cardiocyte of the
guinea pig heart. The ANP-granules (arrows) are found at the pole
of the nucleus (N), among numerous mitochondria (M).
Figure 2. Electron micrograph from right auricle of
guinea pig heart showing a number of granules (arrows)
near the plasma membrane (P).
The number of ANP-granules is influenced by several
physiological conditions: temperature, dehydration and
nutritional condition (Mifune et al., 1993; Gall et al., 1990;
Toyoshima et al., 1996; Penner et al., 1990; Nakayama
et al., 1984; Takayanagi et al., 1985). A reduction of
temperature from 37 to 27 degrees C caused a decrease
of ANP release by 64% in the rat heart. The number of
granules in the cardiocyte increased during dehydration
2536
Afr. J. Biotechnol.
Figure 3. Immunocytochemical staining of ANP-granules in the
right atrium (arrows). No immunocytochemical reaction was
observed outside the granules. Observe the presence of
numerous Protein-A Gold (10 nm), particles inside the granules.
It was demonstrated that there were significant differrences in granule diameter among the regions of the
atrial-auricular complex. The average diameter of the
granules in the right atrium was larger than in the other
regions. The distribution of the granules diameter from
the right atrium was displaced towards larger values
compared to data from the left atrium and auricles.
According to the observations of Mifune et al. (1996), in
various mammalian species the fewer number of granules the cardiocyte had, the smaller the granule size
became, suggesting that the number of granules is possibly related to the granule size. The granule size is therefore possibly determined by the number of granules in the
cardiocytes in the species (Mifune et al., 1996). The ratio
of peptide content in the right atrium was larger than in
the auricles and may indicate a more important endocrine
role in right atrium than in left atrium and auricles.
The size of ANP-granules is also influenced by various
factors. It was small in the presence of water depletion
(Gall et al., 1990). ANP-granules become smaller during
rapid synthesis, especially in spontaneously hypertensive
rats (Takayanagi et al., 1985). It was reported that the
enhancement of ANP synthesis in the cells reduced the
granule size, or ANP-granules became smaller with a
concomitant decrease in the levels of atrial ANP mRNA
and plasma ANP in the course of the down regulation
(Mifune et al., 1991, 1992) According to these authors,
these findings suggest no relationship between the granule size and the ability of ANP synthesis and secretion.
ANP SECRETION
Figure 4. Immunocytochemical staining of ANP-granules in
the right auricle. Several Protein – A Gold particles can be
seen inside the granules (arrows).
(Gall et al., 1990; Toyoshima et al., 1996), but decreased
after Na loading (Penner et al., 1990). Conversely, both
the plasma ANP and ANP mRNA levels increased after
loading (Nakayama et al., 1984; Takayanagi et al., 1985).
These findings suggest that numerical changes in ANPgranules are closely associated with their synthesis and
secretion in the cardiocytes, and that the synthetic and
secretory ability is enhanced in the cell with fewer
granules (Mifune et al., 1996), i. e., in the left atrium and
auricles. Relatively low production of the ANP and/or a
rapid secretion, with the consequent lower storage of the
peptide in these regions, may be the reason.
Since the discovery of ANP more than 20 years ago,
numerous studies have focused on the mechanisms
regulating ANP secretion. Specific atrial granules closeness to the sarcolema, the granule membrane fusion to
the sarcolema and the presence of secretion-like amorphous material present in the endomysium and in the
sub-endocardial space suggest an exocytosis (Figure 5)
process (Gilloteaux et al., 1991). These observations
complement previous descriptions (Imada et al., 1988;
Needleman et al., 1989), demonstrating that the ANP is
eliminated through the atrial myocytes, via exocytosis.
Those authors suggested that the ANP would be released by the emiocytosis process, in which after diffusion
through the sub-endocardial and sub-epicardial spaces
would then be transported through the endocardium,
epicardium and endothelium from vessels to the blood by
means of an endocytosis mechanism, mediated by
receptors. They also suggest that the atrium endothelial
layer can play an important role in the transport control, in
the pro-hormone activation and in the ANP release into
blood stream. It was observed in the rat, that endothelin1, a potent vasoconstrictor produced by endothelial cells,
stimulates the secretion of the ANP by a direct mechani-
Gama and Rodrigues de Souza
Figure 5. Electron micrograph of an atrial cardiocyte. Observe
an empty granule (arrowhead) and a partially empty one (double
arrow). Amorphous material, apparently derived from the
granules, can be observed in the extra-cellular space (arrow).
sm not through a hemodynamic change (Horio et al.,
1993). The dramatic increase in ANP release produced
by cardiac ischemia appears to be mediated in part by
endothelin. Nitric oxide, an important vasodilator, is also
produced by endothelial cells and inhibits ANP secretion
through cyclic GMP as an intracellular messenger (Dietz,
2005).
ANP FUNCTION
ANP is a peptide hormone that causes diuresis, natriuresis, vasodilatation, reduction of plasma renin-aldosterone concentration and depression of blood pressure (De
Bold et al., 1981; Cantin and Genest, 1985; Toyoshima et
al., 1996; Mifune et al., 2004; Cea, 2005). An antiinflammatory ANP potential and a pro-apoptotic action in
rat endothelial cells have also been described (Klinger et
al., 2005). Studies carried out in humans (Tanaka et al.,
1986) and in rabbits (Cho et al., 1991), assessed the
atrial pressure, atrial wall dilatation and contraction
frequency, and suggested that the major stimulus for
ANP release is the frequency increase of cardiomyocytes
shortening. However, an increase of atrial contraction
frequency from 300 to 500/min, in the rat, did not cause a
significant change in the ANP release (Katoh et al.,
1990). According to Katoh et al. (1990), Schiebinger and
Greening (1992), Seul et al. (1992); De Bold et al. (2001)
and Dietz (2005) the main physiological stimulus for
increased ANP release is the atrial muscle stretch, which
normally occurs when extra cellular fluid volume or blood
volume is elevated. It was showed (Katoh et al., 1990)
that an elevation of left atrial filling pressure of working
hearts from 8 to 18 and 28 cm H2O was associated with
pressure dependent, and reversible increase of the ANP
2537
release.
The morphofunctional findings with respect to the
natriuretic peptides from the atria and auricles are becoming very useful, since an increasing concern has been
observed in studies related to cardiac surgeries (Cox et
al., 1991; McCarthy et al., 1993; Yoshihara et al., 1998)
regarding the acute and chronic effects of the ANP release reduction and consequently abnormalities in the
renal function related to body fluid control arising from
cardiac surgeries where removal of the auricles is carried
out. Furthermore, a number of clinical trials suggest that
application of these peptides may represent a new
pharmacological tool in the treatment or prevention of
diseases like acute renal failure or cardiac remodeling in
acute myocardial infarction and congestive heart failure
(De Bold and De Bold, 2005; Michels and Tarnow, 2001).
Elevated ANP and BNP levels may serve as an early
warning system to help to identity patients at high risk for
cardiac events. The clinical applications of natriuretic
peptides are expanding rapidly. Recent basic science
and clinical research findings continue to improve our
understanding of this peptide system and guide use of
ANP and BNP as biomarkers and as therapeutic agents
(Silver, 2006).
CONCLUSIONS
In conclusion, the ANP is present in all the granules from
the four regions of the atria and auricles and the right
atrium is the region with the largest number of granules.
The small granules are, by far, the most numerous in the
four regions. It is possible that the wall of the right atrium
is the most sensitive region to principal mechanism for
the liberation of the ANP granules that is the distension of
the wall musculature (De Bold and De Bold, 2005),
because it is the area where the largest number of
granules and those with the biggest size were
concentrated. The significantly greater number of right
atrial ANP positive granules suggest a greater volume
capacity for the right atrial ANP- positive granules as
compared to the other regions of the heart. This may
indicate that ANP is secreted primarily from the right
atrium and to a lesser extent from the left atrium and
auricles and that both atrial and auricular ANP are closely
related in chemical nature and immunocytochemical
characteristics.
ACKNOWLEDGEMENT
RRS is career investigator of CNPq (National Council of
Research).
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